Natural proteins are made up of linear chains of amino acid residues linked by amide bonds. The polypeptide chain is flexible and has virtually complete conformational freedom. Despite this freedom, natural polypeptides adopt tightly packed, highly ordered folded structures such as alpha-helices, beta-sheets and beta-turns, i.e., form non-random secondary structures. The energy balance between folded and unfolded states of polypeptides is thought to be regulated by conformational entropy factors. One such factor, the hydrophobic effect, is now thought to be the major driving force in protein folding. Dill, K. A., Biochemistry, 24:1501 (1985).
When two or more hydrophobic groups (initially solvated by a highly ordered water shell) clump together in the interior of a protein, the increase in the solvent entropy is more than compensated for by the loss of conformational entropy accompanying this event. The interaction of the polypeptide with the solvent surroundings causes non-polar side chains to cluster together in the interior and the hydrophilic groups to remain largely at the protein surface. According to one theory, the hydrophilic interactions force rapid collapse of the polypeptide to a compact state called the "molten globule", which then rearranges the final structure. See. e.g., Pain, R., Trends Biochem. Sci. Pers. Ed., 12:309 (1987); Dolgikh et al., FEBS Lett., 136.311 (1981); and Kuwajima et al., FEBS Lett., 221:115 (1987).
Folded structures such as alpha helices and beta sheets are maintained by an extensive hydrogen bond network. Richardson, J. S., Adv. Protein Chem., 34:167 (1981). Although the contribution of a single such hydrogen bond is typically very small, the cooperative formation of hydrogen bonds, particularly in the interior of a protein, likely plays an important role in folding and stability. Other forces contributing to the stability of folded polypeptides are electrostatic forces, medium and long-range ion-pairing interactions, helix-dipole interactions, covalent forces such as disulfide bonding and the nature of C- and N-termini amino acid residues. See, e.g., Barlow et al., J. Molec. Biol., 168:867 (1983); Perutz, M. F., Science, 201:1187 (1978); Richardson et al., Science, 240:1648 (1988); and Presta et al., Science, 240:1632 (1988). Dispersion forces and van der Waal's forces are likely less important in this regard.
Despite the accumulation of a large body of information about folded protein structures, the de novo design of artificial polypeptides having a defined conformation remains an elusive task. The major obstacle in the design of such polypeptides from linear polypeptide sequences is the lack of any comprehensive understanding of how the one-dimensional sequence information directs the formation of a discrete three-dimensional state (topology) of a polypeptide.
Two types of approaches, biometric and nonlinear, have been recently reported and utilized for the de novo design of polypeptides. See, e.g., Richardson et al., Trends Biochem. Sci., 14:304 (1989); and Mutter et al., Agnew. Chem. Int. Ed. Engl., 5:535 (1989). The biometric design approach attempts to model the natural shape and structure of proteins by designing linear peptide sequences that are predicted to fold into a desired topology. For example, recombinant DNA technologies have been used to design and construct a synthetic polypeptide having an anti-parallel tetra-helical topology. See, e.g., Hecht et al., Science, 249:884 (1990); and Regan et al., Science, 241:976 (1988). A tri-helical peptide has recently been described. Lieberman et al., J. Am. Chem. Soc., 113:1470 (1991).
Other attempts to design and construct synthetic polypeptides having a desired topology have been hampered by a number of problems. A beta-barrel protein called "b-bellin" has been designed but its structural characteristics remain unknown because of solubility problems. Richardson et al., Trends Biochem. Sci., 14:304 (1989). Similar solubility problems have been encountered with synthetic polypeptides having combined beta- and alpha-helical topologies.
Some problems have been avoided by the use of non-linear design principles. Using a template-assisted approach, several alpha-helical and beta-strand polypeptides comprising identical amphiphilic peptides have been designed. See, e.g., Sasaki et al., J. Am. Chem. Soc., 111:380 (1989); and Hahn et al., Science, 248:1544 (1990). The topology of these polypeptides, however, has not yet been fully elucidated.
One approach for the construction of artificial polypeptides having a defined topology is to develop self-organizing molecular processes by which small peptides can be assembled into large and topologically predetermined polypeptide tertiary structures.
The present invention provides a polyvalent metal ion-assisted self-assembly process for the construction of artificial polypeptides in which the overall topology of the polypeptide is manipulated by exploiting the interaction of a polyvalent metal ion with an appropriately designed metal ion binding site.